Leguminous plants, such as soybean, form a symbiotic relationship with dinitrogen (N2) fixing bacteria commonly called “rhizobia” that live within nodules on the roots, forming an intimate inter-kingdom association of the two organisms to reduce (“fix”) dinitrogen gas in the atmosphere into a form which the plants can use as a nitrogen source. As a result, well-nodulated plants do not require extensive nitrogen fertilization. The slow-growing bacteria forming symbioses with certain legumes including soybean belong to the family Bradyrhizobiaceae. These microorganisms are a diverse group of gram-negative, nonspore-forming, rod-shaped, aerobic alpha-proteo bacteria.
Originally, rhizobia were classified as a single genus. More recent classifications, however, have placed legume-nodulating nitrogen-fixing rhizobia into six genera, belonging to a four families, within the new order Rhizobiales. These distinct taxonomic groups are based upon sequence similarities of 16S rRNA. Current taxonomic divisions of rhizobia have placed several clusters of rhizobia in four families together within the alpha-proteo bacteria based upon these sequence similarities. Among the most common genera within rhizobia are: Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium, and Bradyrhizobium. 
The bacteria infect the root, forming nodules where biological N2 fixation occurs that supplies 40 to 85% of the soybean's nitrogen requirements. Nitrogen fixation commences about two-to-three weeks after the infection process begins and is indicated by large, nearly spherical yet somewhat irregularly shaped nodules having a textured surface with localized areas of pinkish red interior color due to the presence of leghemoglobin. Early in the growing season, nodules are clustered near the root crown. Later, the younger nodules located on secondary roots become more important in N2 fixation activity. Nodules must be present for significant N2 fixation to occur as they provide a protected ecological niche for the nitrogen-fixing bacteroids, a differentiated form of varying viability depending on the bacterial species.
Not all nodules are effective, however. Effectiveness of nodules is reflected in the ability of the bacteria within nodules to fix dinitrogen. Effective nodules are those nodules formed on legume roots that have the ability to fix (reduce) N2 symbiotically at high rates relative to a recognized superior rhizobial strain which serves as a standard strain, such as strain USDA 110 for B. japonicum infecting soybean. Kuykendall and Elkan (1976) described the derivation of strain 110 substrains varying many fold in symbiotic nitrogen fixation ability and also differing qualitatively and/or quantitatively in ability to utilize simple 5 and 6 carbon polyols and hexose sugars.
Glyphosate [N-(phosphonomethyl)glycine] is the active ingredient in the non-selective herbicide Roundup® (Monsanto Co., St. Louis, Mo. 63167). Advances in biotechnology have resulted in glyphosate resistant or tolerant (GR) soybean cultivars, providing an effective broad-spectrum post-emergence weed-control option. Glyphosate competitively inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Commercial glyphosate resistant or tolerant (GR) soybean expresses an EPSPS that is resistant or tolerant to glyphosate.
Glyphosate is not readily degraded in soybean and concentrates in metabolic sinks, such as young roots and developing and mature nodules. A single foliar application of glyphosate at 0.5 kg ha−1 can result in concentrations up to 0.3 mM in bulk root tissue of susceptible plant species. Higher glyphosate use rates or repeated applications may result in even greater concentrations, especially in the stronger metabolic sinks such as soybean root nodules as compared to the bulk root system.
The use of glyphosate as a herbicidal chemical to control weeds in the cultivation of specially resistant crop plants, such as soybean, has been historically viewed as largely innocuous with regards to symbiotic nitrogen fixation in legumes including soybeans. However, detrimental effects may occur since, although EPSPS in GR soybean is resistant or tolerant to glyphosate, strains of the N2 fixing microsymbionts, such as Bradyrhizobium japonicum, have a sensitive form of the enzyme. Researchers have concluded that glyphosphate herbicide may show enough cytotoxicity, particularly to certain agronomical-elite, seed-borne inoculant rhizobia, to reduce their viability and thus lower their ability to contribute nitrogen to the planted crop (Zablotowicz and Reddy, 2004)
While applications of environmentally unwise and expensive chemical fertilizer can offset inoculation failure, this practice is not sustainable and less costly and more environmentally friendly alternatives are needed. Genetically engineered bacterial strains may carry undesirable, introduced antibiotic resistance gene(s), for example kanamycin resistance. It is therefore an unmet advantage of the prior art to provide biologically effective, herbicide resistant bacterial strains via direct mutant selection.